We have previously demonstrated that the Sle1 gene cluster is a key element in initiating the autoimmune cascade that leads to fatal lupus in the NZM2410 mouse. Our positional cloning analysis of Sle1b identified a seven-gene cluster of CD2 family genes (SF gene cluster) as causative for a breach in immune tolerance to nuclear antigens. The SF cluster contains CD48, CD84, 2B4, SLAM, Ly9, Lyl08, and CSI. A variety of studies indicate that these genes modulate the activation thresholds and effector functions of several immune cell lineages. Although our genetic analyses clearly implicate the SF gene cluster with systemic autoimmunity; the mechanism by which these genes mediate disease is unknown. The strongest single gene candidate in the SF cluster is Ly108, in which the Ly108-1 isoform is constitutively upregulated in B6.Sle1b B cells. However, autoimmunity may not be caused by a single gene, but rather by the combined effects of multiple polymorphic alleles in the SF gene cluster. Here we propose to characterize the functional properties of these candidate genes in vitro and in vivo and use genetic manipulation to directly test the ability of alleles of these genes to breach immune tolerance. We have four specific aims: 1) To assess the expression of SF cluster genes in vivo. A detailed characterization of relevant genes in this cluster will require the production of monoclonal antibodies and transgenic mice with expression constructs. 2) To define the role of Ly108 in lymphocyte function. We will assess the role of Ly108 in T and B cell proliferation, activation, tolerance and effector functions. These studies will be performed using in-vitro and in-vivo assays with B6 and B6.Sle1b mice. 3) To assess the ability of Ly108 to breach tolerance to nuclear antigens in vivo. We have produced a series of constructs expressing specific isoforms of Ly108 to determine whether over expression of Ly108-1 in B cells will breach tolerance to nuclear antigens in B6 mice, while over-expression of Ly108-2 will suppress autoimmunity in B6.Sle1b. We will also produce similar constructs expressing Ly108 anti-sense mRNA (RNAi) to assess the impact of disrupting Ly108 expression on the development of the immune system and immune responsiveness. 4) To assess the functional properties of the SF cluster in vivo. To develop a system that will allow an analysis of the interactions among SF members, we will integrate flanking LoxP sites and delete the SF cluster in 129 ES cells. SF cluster null mice, combined with a BAC rescue strategy with individual members of the SF family, will allow an assessment of function of this gene cluster in vivo.